Abstract

All-trans-retinoic acid (ATRA) has significantly improved
the treatment results in acute promyelocytic leukemia (M3). In non-M3
acute myeloid leukemia (AML), the effects are less clear, and there is
a pronounced heterogeneity in the sensitivity to the growth-inhibitory
effects of retinoids in leukemic cells from different non-M3 AML
patients. Retinoids exert their effects through a number of nuclear
receptors [retinoic acid receptors (RARs) and retinoid X receptors
(RXRs)]. In this study, we determined the expression of RARα,
RARβ, RARγ, and RXRα by real-time PCR in four cell lines and in
blast cells from patients with non-M3 AML before and after ATRA
incubation. All four receptors were expressed in cells from all 18
tested patient samples and in four myeloid cell lines. In the majority
of the patient samples as well as in the cell lines, there was a
pattern of high expression of RARα and RXRα and low expression of
RARβ and RARγ. There was no correlation between the basal
expression of any of the retinoid receptors and sensitivity to ATRA. A
24-h exposure to ATRA increased the expression of RARα, RARβ,
RARγ, and RXRα in 46%, 77%, 30%, and 38% of the samples,
respectively. The mean increase in receptor expression was most
pronounced for RARβ and RXRα. There was a significant correlation
between an increase in RARβ expression in response to ATRA and
sensitivity to ATRA (P < 0.014). No such
correlations were found for RARα, RARγ, and RXRα. The expression
of the monocytoid marker CD14 was significantly correlated with
increased expression of RARα (P = 0.03). We
conclude that RARα, RARβ, RARγ, and RXRα are expressed in
non-M3 AML blast cells and that ATRA-induced expression of RARβ may
be a marker for retinoid sensitivity.

INTRODUCTION

Retinoids are important regulators of normal cell growth and
differentiation (1, 2)
, but they also have profound
effects on tumor cell growth in various malignant tumor species both
in vitro and in vivo(3, 4, 5, 6, 7, 8, 9)
. The
cellular effects of the retinoids are mediated through two types of
intracellular receptors belonging to the steroid receptor superfamily,
RARs3
and RXRs, each consisting of α, β, and γ isoforms. These
receptors form RAR/RXR or RXR/RXR dimers that act directly on retinoid
acid response elements within the promotor region of certain genes
(10, 11, 12)
. The retinoid receptors are believed to mediate
the physiological as well as the pharmacological effects of retinoids.
The role of each subtype of the receptors may differ (11, 13)
, but overexpression of either RARα, RARβ, RARγ, or
RXRα in transfected myeloid progenitor cells renders these cells
sensitive to the growth-inhibitory effects of retinoids
(14)
.

In APL (or M3 according to the FAB classification), ATRA is highly
effective and has improved the complete remission rate as well as
relapse-free and overall survival (15, 16, 17, 18, 19)
. These results
have encouraged studies of treatment with retinoids in other
malignancies. In non-M3 AML blast cells, the effect of retinoids is
less pronounced compared to that in APL cells, but even so, leukemic
cells from up to 70% of non-M3 AML patients are sensitive to ATRA or
9-cis-RA (20, 21)
. Except for the heterogeneity
in the response to retinoids between non-M3 AML cells from different
patients, the sensitivity also varies between different tumor
species. New and more receptor-specific ligands are continuously
synthesized and studied to make treatment with retinoids more effective
(22, 23, 24, 25)
.

In several tumor species, attempts have been made to predict retinoid
sensitivity in tumor cells by correlating the expression of the
retinoid receptors to the growth-inhibitory effects of retinoids. In
some studies, the up-regulation of RARβ in response to ATRA exposure
has been shown to correlate with retinoid sensitivity both in
vitro and in vivo in renal cell cancer
(26, 27, 28, 29)
. Fitzgerald et al.(30)
also found a correlation between the basal expression of RARα and
sensitivity to retinoids in breast cancer cells.

To investigate the relationship between receptor expression and
retinoid sensitivity in non-M3 AML blast cells, we determined the mRNA
expression of RARα, RARβ, RARγ, and RXRα by real-time PCR in
non-M3 AML blast cells before and after exposure to ATRA. The results
were then correlated with the antitumoral effects of ATRA in
vitro and with several disease characteristics. RARα, RARβ,
RARγ, and RXRα were expressed in all myeloid cell lines and in
leukemic cells from all patients with non-M3 AML. There was no
correlation between the basal receptor expression of any of the
receptors and the growth-inhibitory effects of ATRA. However,
ATRA-induced up-regulation of RARβ was significantly correlated with
ATRA sensitivity. Moreover, we found a significant correlation between
RARα expression and CD14 positivity.

MATERIALS AND METHODS

Cell Lines and Patient cells.

The HL-60, K562, and KG1a cell lines were obtained from the American
Type Culture Collection (Manassas, VA). A multidrug-resistant,
P-glycoprotein-expressing, ATRA-resistant HL-60 cell variant,
HL-60R, has been obtained by selection for resistance to doxorubicin as
described previously by Jönsson et al.(31)
.

Leukemic cells from 18 non-M3 AML patients were collected from the bone
marrow at the time of the diagnosis. Blast cells were isolated by the
density gradient with Ficoll separation (Lymphoprep Nyregaard end Co
AS, Oslo, Norway). The mean age of the patients was 66 years (age
range, 37–83 years); 10 patients were male, and 8 patients were
female. Three patients were classified as M1, three patients were
classified as M2, one patient was classified as M3 [without
t(15;17)], six patients were classified as M4, one patient was
classified as M5b, one patient was classified as M6, and three patients
were not classified. After cell separation, cells were washed twice in
RPMI 1640 and resuspended in RPMI 1640 with 10% FCS (1.0 ×
106 cells/ml). The cells were then either
analyzed immediately or cryopreserved in PBS with 20% FCS and 10%
DMSO in vapor phase nitrogen. For determination of ATRA-induced change
in receptor expression, a proportion of the blast cells were incubated
in RPMI 1640 with 10% FCS and 1 μm ATRA for 24 h
(5% CO2; 37°C) before the receptor analysis.

Assessment of Viability.

For assessment of retinoid sensitivity, patient cells were incubated in
RPMI 1640 with 10% FCS with or without 1 μm ATRA or
9-cis-RA (Roche AB, Stockholm, Sweden). Cells were
incubated in a humidified incubator at 5% CO2
and 37°C for 96 h. All incubations were performed on fresh
cells. The cell viability was assessed by a bioluminescence assay
measuring ATP content as described previously (20, 32)
.
The assay was performed automatically in a Bio Orbit (Turku,
Finland) photometer. The AMR and the ATP standard used were both
supplied by Bio Orbita. The ATP standard was reconstituted in 10
ml of distilled water, giving a 10 μm solution.
AMR was reconstituted with 5 ml of Tris-EDTA buffer [100
mm Tris and 2 mm EDTA (pH
7.75)]. Twenty μl of the cell sample were put in the cuvette.
Automatically, 200 μl of AMR were dispensed in a cuvette placed in
the photometer, and the resulting light emission was measured. ATP
standard was automatically added (10 μl), and the emitted light was
measured. The amount of ATP was calculated with correction for the
blanks. The results were given as nanomoles of ATP/sample. The
percentage of ATP in a sample compared to drug-free control was then
calculated. The result of each exposure represents a mean of the two
parallel exposures. To ensure the viability of controls, this was
examined after 24 h of incubation and at the end of the incubation
(after 96 h). All controls samples had >30% viable cells
compared with day 1.

Immunophenotyping and Cytogenetic Analysis.

Before immunophenotyping, erythrocytes were lysed in
NH4Cl for 8 min. Cells were then incubated with
FITC-, phycoerytrin-, or PeRCP-conjugated antibodies [Becton
Dickinson (San Jose, CA) and Dakopatts (Glostrup, Denmark;
RPEcy5 conjugate)] for 15 min using a routine panel of
antibodies for fluorescence-activated cell-sorting analysis of de
novo AML. The samples were analyzed by three-color
immunofluorescence in a FACSscan flow cytometer (Becton
Dickinson). A cutoff limit of 20% was set to define a sample as being
negative or positive. In all samples defined as positive, 38–100% of
the cells were positive, with the majority of the samples showing>
50% positive cells. No negative sample showed more than 14%
positive cells.

Cytogenetic analyses were performed at the time of the diagnosis.
Either G banding or Q banding was made on the bone marrow material
after a 24-h incubation. The chromosomes and the aberrations were
classified according to the ISCN 1995.

Real-time PCR.

Total RNA was isolated from the cells of AML patients using Trizol
(Life Technologies Inc., Gaithersburg, MD) and transcribed into cDNA as
described previously (33)
. The PCR reaction mixture
included 10 mm Tris-HCl (pH 8.3), 50 mm KCl,
5.0 mm MgCl2, 0.4 mm each
deoxynucleotide triphosphate, and 1.25 units of AmpliTaq Gold DNA
polymerase (Perkin-Elmer, Sundbyberg, Sweden). Primers and fluorogenic
probes were added to a final concentration of 0.4 and 0.2μ
m, respectively (33)
. Total PCR volume was
25 μl, including 1 μl of the reverse transcription reaction that
equals 50 ng of total RNA. Duplicate reaction tubes were set up
for each sample and transcript under investigation. The tubes were
placed in an ABI Prism 7700 System programmed for 40 sequential cycles,
each comprising heating to 94°C for 15 s followed by 60°C for
30 s. A standard curve was generated by amplifying known amounts
of a PCR product at the same time as the samples. PCR primers and
TaqMan Fluorogenic DNA probes were as follows (sequences are given in
5′-3′ orientation): (a) β-actin, CTGGCTGCTGACCGAGG
(forward primer), GAAGGTCTCAAACATGATCTGGGT (reverse primer), and
CCTGAACCCCAAGGCCAACCG (TaqMan probe); (b) RARα,
CTCCATGCCGCCTCTCAT (forward primer), CGGCTGTCCGCTCAGAGT (reverse
primer), and CAGGCCCTCTGAGTTCTCCAACATTTCCT (TaqMan probe);
(c) RARβ, AGCCTACGTGCCAAAAAAGG (forward primer),
TCTAGGTGTGGAGGCAAATGG (reverse primer), and
AGAAAAGTCCACCCAACTCCATCAAACTCTG (TaqMan probe); (d)
RARγ, TGTGCGAAATGACCGGAAC (forward primer),
CTAACTGAGGGCTCAGCTCATAGC (reverse primer), and
CAGGTGACCCTTCTTCCTTCACCTCTTTCTTC (TaqMan probe); and (e)
RXRα, CCCTGTCACCAACATTTGCC (forward primer), AGAAGTGTGGGATCCGCTTG
(reverse primer), and AGCAGCCGACAAACAGCTTTTCACCC (TaqMan probe).

Statistics.

Mean values with the SE for each retinoid receptor were calculated from
the molar ratios of each receptor (receptor mRNA:β-actin mRNA). The
differences in the receptor expression between samples with respect to
their CD status were evaluated with Student’s unpaired
t test. The correlation between ATRA-induced effect on
receptor expression and ATRA sensitivity was determined by Fisher’s
exact test.

RESULTS

Expression of Retinoid Receptors in Four Myeloid Cell Lines.

The expression of retinoid receptors and the housekeeping geneβ
-actin was investigated in the four myeloid cell lines
(HL-60, HL-60R, KG1a, and K562), and the receptor expression is
expressed as a molar ratio (receptor:β-actin). Receptor transcripts
of all receptors were found in all four cell lines; however, the
expression varied significantly (Fig. 1)⇓
. RARα and RXRα expression was 100-1000-fold higher than that of
RARβ and RARγ, with the exception of the K562 cells, in which
RARα and RXRα expression was only10-fold higher. RARα was
most abundant in HL-60 cells, and expression of RARα was considerably
lower in the multidrug-resistant HL-60R cells. Compared to the other
cell lines, RARp and RXRα expression was high in the K562
cells, and RARγ expression was somewhat higher in HL-60R and K562
cells.

Expression of Retinoid Receptors in AML Patient Cells.

Expression of all four receptors (RARα, RARβ, RARγ, and RXRα)
could be detected in all of the 18 patient samples; however, as in the
cell lines, the expression varied considerably (Fig. 2)⇓
. The pattern of retinoid receptor expression was also similar to that
seen in the cell lines, but with less pronounced differences in
expression between the four receptors. As in the cell lines, the level
of RARα and RXRα was higher than that of RARβ and RARγ. This
pattern was seen in 15 of the 18 patients samples, but 3 of the patient
samples exhibited another receptor profile with high expression of both
RARβ and RARγ in 1 sample and low RXRα expression in 2 samples.
RXRα showed the highest level of average expression of all receptors,
and RARβ showed the lowest level of average expression of all
receptors, somewhat lower than that for RARγ. The expression in
cryopreserved cells did not differ from that in fresh cells.

Fifteen of the 18 patients were classified according to the FAB system.
All subgroups exhibited a similar pattern of expression, except for the
patient with M6 who presented with high RARβ and RARγ expression.
There was also a higher expression of RARα in samples from patients
with AML M4, but this difference was not statistically significant.

The correlation between the expression of CD markers and receptor
expression was also analyzed. The expression was compared in cells that
had been defined as either positive or negative for CD34, HLA-DR, CD14,
CD15, CD13, CD33, and CD56. However, as only one and two of the samples
were negative for CD13 and CD33, respectively, these CD markers were
excluded from this analysis. For the remaining CD markers, no less then
four cases were either negative or positive. The comparison
between receptor expression and the expression of HLA-DR, CD15, and
CD56 revealed no differences. For CD34-positive cells, the mean
expression of RARα, RARγ, and RXRα tended to be lower than that
seen in CD34-negative cells, but the difference was close to
significance only for RARγ (P = 0.08; Table 1⇓
). The only statistically significant difference was the increased
RARα expression in the CD14-positive cells (P =
0.03). Mean receptor expression and Ps for each receptor
with regard to their CD34 and CD14 status are presented in Table 1⇓
.

Cytogenetic analyses could be assessed in 11 of the patients. In only
one case did the chromosome aberration interfere with the locus of any
of the genes coding for the four retinoid receptors. This case showed a
monosomy of chromosomes 3, 9, and 12, corresponding to the gene
locations of RARβ, RXRα, and RARγ, respectively. Although the
basal expression of the three receptors was not decreased in this
sample, the ability to up-regulate RARβ, RXRα, and RARγ
expression in response to ATRA was impaired compared with the other
patient samples. Meanwhile, the ATRA-induced expression of RARα
expression was not impaired compared with the average
expression. The changes in receptor expression after ATRA
incubation in this single case were (expressed as the difference
between basal and ATRA-induced expression) +35% for RARβ (average,+
330%), −70% for RARγ (average, +48%), +150% for RXRα
(average, +360%), and +210% for RARα (average, +12%).

Correlation between Expression of Retinoid Receptors and
Sensitivity to ATRA.

The average viability of the patient cells after 96 h of
incubation in 1 μm ATRA was 78% (range, 10–119%)
compared with that of unexposed cells. No correlation between the basal
expression of the retinoid receptor and ATRA sensitivity was seen for
any of the receptors, and correlation coefficients between ATRA
sensitivity and receptor expression expressed as molar ratios were
close to 0 for all four receptors. Because any of the investigated
retinoid receptors is a potential mediator of the growth-inhibitory
effects of ATRA, we also examined whether high expression of any of the
receptors in patient cells or a high amount of total receptor mRNA in
patient samples could be correlated with retinoid sensitivity.
However, neither of these comparisons revealed any correlation. In one
patient sample, ATRA increased the growth of leukemic cells, but the
pattern of receptor expression was not different from that of the other
cells.

In 13 of the patients, receptor expression was also examined after a
24-h exposure to 1 μm ATRA. Table 2⇓
shows the effect of the ATRA incubation on the expression of each
receptor in all patient samples. RARβ was up-regulated by ATRA in the
majority of the patient samples (77%). RARα, RARγ, and RXRα were
less commonly up-regulated (38%, 31%, and 46%, respectively).
Although RARβ up-regulation was the most common, the highest
increment in expression was seen for RXRα, showing a 25- and 15-fold
increase in two patient samples.

Correlations were then performed between the growth-inhibitory effects
of ATRA and the ATRA-induced change in receptor expression. We studied
whether ATRA sensitivity, defined as a decrease in viability to <95%
compared with unexposed controls, correlated with the change in
receptor expression after ATRA exposure. These comparisons showed that
an increase in RARβ was significantly correlated with ATRA
sensitivity (P = 0.014), a relationship that could not
be found for any of the other receptors (Table 2)⇓
. Comparing mean
sensitivity to ATRA in samples that did up-regulate receptor expression
with those that did not showed a significant difference for RARβ[
73% versus 103% viability for samples with and without
RARβ up-regulation (P = 0.01)]. All samples that did
not up-regulate RARβ were also ATRA resistant. Correlations between
the retinoid sensitivity and ATRA-induced change in receptor expression
were identical when receptor expression was compared with sensitivity
to 9-cis-RA (significant correlation to RARβ up-regulation
but not to any change of RARα, RARγ, or RXRα; data not shown).

DISCUSSION

Since the introduction of ATRA in standard treatment of APL, there
has been a constant search for the role of retinoids in other malignant
diseases. New and more receptor-specific retinoids are continuously
synthesized, and the knowledge about the mechanism of action of
retinoids has grown considerably. A problem with retinoid-based
therapies has been that the antitumoral effects vary significantly
between tumor species and between individual patients with the same
diagnosis. Treatment with retinoids in non-M3 AML has attracted
interest in recent years and new randomized trials have been
initiated. A majority of the non-M3 AML blast cells are sensitive to
either ATRA or 9-cis-RA in vitro but with
a considerable proportion of the cells being resistant (20, 21)
. This stresses the need for more knowledge about the
molecular mechanisms of retinoids and the demand for tools to predict
retinoid sensitivity. In this study, we examined the expression of
retinoid receptors before and after ATRA exposure and whether the
expression correlated to retinoid sensitivity in non-M3 AML blast
cells. We also correlated the expression to other characteristics of
the cells.

Retinoid receptor mRNA for RARα, RARβ, RARγ, and RXRα could be
detected in all 18 patient samples and in four myeloid cell lines. In
both the cell lines and the patient cells, expression of RARα and
RXRα was high compared to that of RARβ and RARγ, although the
differences in the patient cells were less pronounced. This
relationship between the expression of the different receptors is
similar to what has been found in other malignant cells (28, 34, 35, 36)
. However, leukemic blast cells from AML patients are
heterogeneous, and we did not examine whether the receptor expression
was altered in different leukemic subpopulations. The presence of
subpopulations with different receptor expression may also explain why
differences in the expression of the receptors (i.e., RARα
and RXRα versus RARβ and RARγ) were less pronounced in
patient cells compared with the cell lines. The different FAB classes
exhibited mainly the same expression pattern, with exception of the
single M6 case. However, no conclusion should be drawn from this single
case.

In this study, we did not examine the expression at protein level.
However, in previous studies on tumor tissues, there has been a good
correlation between retinoid receptor mRNA and protein levels
(34)
. This is also expected because the
T1/2 for the proteins is short
(37)
.

In contrast to the findings by Fitzgerald et al.(30)
, we found no correlation between retinoid sensitivity
and basal receptor expression for any of the receptors on the mRNA
level. This is also in accordance with most previous studies and
reinforces the belief that the level of receptor expression itself does
not determine the response to retinoids but that the cellular response
is instead determined by other coregulating factors on transcriptional
level (38, 39, 40)
.

Retinoids bind to nuclear receptors that form homodimers or
heterodimers and then, together with coregulating factors, act on RA
response elements and regulate the transcription of a variety of genes
(11)
. One of the target genes is RARβ, which can be
activated by the binding of a receptor complex to its promoter region
(41, 42)
. A key role has been attributed to RARβ because
it may act as a tumor suppressor and because the growth-inhibitory
effects of retinoids correlate to the ability to up-regulate RARβ in
response to ATRA exposure in some tumor species (1, 26, 27, 28, 43)
. Based on these previous findings, we also analyzed receptor
expression in cells exposed to 1 μm ATRA for 24 h.
In the patient cells, changes in receptor expression in response to
ATRA were most prominent for RARβ and RXRα. For RARβ, a high
proportion of the samples responded with up-regulation, whereas for
RXRα, the amplitude of the up-regulation was considerable for some
patient samples. Changes for RARα and RARγ were less prominent, and
increased as well as decreased expression was seen. These results
correspond to findings in renal cancer cell lines, melanoma cells, and
myeloid progenitor cells (14, 27, 44)
.

By correlating the ATRA-induced change in receptor expression with ATRA
sensitivity, we found a significant correlation between the sensitivity
to ATRA and up-regulation of RARβ. This correlation was not seen for
any of the other receptors. The finding is consistent with previous
observations in other tumor species but has not been shown for AML.
This clearly suggests a link between the growth-inhibitory effects of
ATRA and RARβ up-regulation in non-M3 AML, and it could offer a
possibility to predict the response to retinoids when RARβ expression
is analyzed in vivo during ATRA treatment. This is currently
being investigated in a clinical trial.

Retinoids usually induce growth inhibition and cause decreased cell
survival but may also promote cell growth (20, 45, 46)
,
and the predominant effect in hematological progenitor cells is
dependent on the maturity of the cell (46, 47)
. It can be
hypothesized that such diverse effects could be caused by changes in
the expression of retinoid receptors during maturation and
differentiation. There has also been one report about increased
expression of RXRα on monocytoid differentiation (48)
.
To study the relationship between differentiation of the leukemic blast
cells and expression of retinoid receptors, we compared the receptor
expression to the expression of some hematological differentiation
markers. Although the number of samples was limited, we found an
increase in RARα expression in samples expressing the monocytoid
differentiation marker CD14. CD34-positive cells tended to have a lower
expression of three of the receptors, but the difference was only close
to significance for RARγ. For the other CD markers, either there were
too few negative samples to be analyzed (CD13 and CD33) or the
comparison failed to show any differences (HLA-DR, CD15, and CD56). The
increased RARα expression in CD14-positive cells may also correspond
to the tendency toward increased RARα expression that was found in
AML samples with monocytoid differentiation (M4). However, because
leukemic cells often express aberrant phenotypes, it is difficult to
draw any conclusion regarding retinoid receptor expression during
normal hematopoietic maturation and differentiation. We are currently
investigating receptor expression in different subsets of normal
hematopoetic cells.

Loss of heterozygosity at chromosome 3p24 has been found in lung cancer
cells, and this has provoked hypotheses regarding the role of RARβ in
the carcinogenesis of that disease (34)
. Only one patient
sample carried chromosome aberrations that interfered with the locus of
any of the retinoid receptor genes, and this patient presented with
monosomy of three chromosomes (chromosomes 3, 9, and 12), representing
the locus for the genes coding for RARβ, RARγ, and RXRα. Although
the basal expression of the affected receptors was intact in these
cells, the ability to up-regulate receptor expression in response to
ATRA was impaired. However, because this represents the result in only
one patient sample, it is difficult to draw further conclusions.

In summary, this is the first time, to our knowledge, that retinoid
receptor expression has been analyzed with a real-time PCR technique in
myeloid non-M3 leukemic cells. The results show that RARα, RARβ,
RARγ, and RXRα are expressed to variable degree in these cells and
that ATRA sensitivity correlates with ATRA-induced up-regulation of
RARβ expression but not with the basal expression of any of the
receptors. Retinoid protocols for non-M3 AML patients are currently
under investigation, and the ability to up-regulate RARβ may be
useful as a predictive test during treatment with retinoids.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.